Inhibition of betaarrestin mediated effects prolongs and potentiates opioid receptor-mediated analgesia

ABSTRACT

The present invention provides a βarrestin knockout mouse useful for screening compounds for efficacy in controlling pain, methods of controlling pain in subjects by inhibiting binding of βarrestin to phosphorylated μ opioid receptors, and methods of screening a compound for activity in potentiating μ opioid receptor agonist activity (e.g., morphine activity) by determining whether or not said compound inhibits βarrestin binding to a phosphorylated μ opioid receptor.

[0001] This invention was made with Government support under NIH grantnumbers NS 19576 and HL16037. The Government has certain rights to thisinvention.

FIELD OF THE INVENTION

[0002] The present invention concerns transgenic mice useful forscreening compounds for their ability to control pain, methods ofcontrolling pain in subjects in need thereof, methods of screening acompound for activity in controlling pain, and/or screening compoundsfor opioid receptor agonist activity.

BACKGROUND OF THE INVENTION

[0003] G protein coupled receptors (GPCRs) have important roles inmediating fundamental physiological processes such as vision, olfaction,cardiovascular function, and pain perception. Cellular communicationthrough GPCRs requires the coordination of processes governing receptoractivation, desensitization, and resensitization. However, the relativecontribution of desensitization mechanisms to the overall homeostaticprocess still remains largely unexplored in vivo. GPCR kinases (GRKs)act to phosphorylate activated receptors and promote their interactionwith βarrestins. This, in turn, prevents further coupling with Gproteins and disrupts normal activation of the second messengersignaling cascade. By this mechanism, GRKs and βarrestins can act todampen GPCR signaling, thereby leading to desensitization of thereceptor (S. Ferguson, et al., Annu Rev Biochem 67, 653 (1998)). Atleast six GRKs (GRK1-6) and four arrestins (visual and cone arrestin,βarrestin-1 and -2) have been discovered; however, the functionalsignificance of such redundancy is unclear.

[0004] Overexpression or inactivation of certain GRKs leads tomodulation of receptor responsiveness (W. Koch, et al., Science 268,1350 (1995); H. Rockman et al., Proc Natl Acad Sci USA 93, 9954 (1996);D. Choi et al. J Biol Chem 272, 17223 (1997); G. Iaccarino et al., Am JPhysiol 275, H1298 (1998); K. Peppel, et al., J Biol Chem 272, 25425(1997); H. Rockman, et al., J Biol Chem 273, 18180 (1998). J. Walker etal., Am J Physiol 276, R1214 (1999)). In addition, mice that aredeficient in βarrestin-1 display increased cardiac contractility inresponse to β-adrenergic receptor agonists (D. Conner et al., Circ Res81, 1021 (1997)).

SUMMARY OF THE INVENTION

[0005] Pain perception (nociception) is mediated by a cascade of eventsfrom the point of the stimulus to integrative circuits in the brain.Nociception involves signals that are mediated by several classes ofreceptors and signal transduction mechanisms such as GPCRs for substanceP, opioid peptides, etc. and ion channels such as NMDA receptors.Antinociception has been known for more than 1000 years to be induced bythe alkaloid compound, morphine, which functions as an agonist at the μopioid receptor. The activity of agonists for signaling through GPCRs isusually limited by cellular mechanisms that dampen the signal of theagonist, a process referred to as desensitization. These mechanismsinclude phosphorylation of agonist-activated receptors by specificreceptor kinases called GRKs followed by the interaction of thephosphorylated GPCR with any of the members of the arrestin family ofproteins. Morphine-mediated antinociception is known to wane with time,however the contribution of the desensitization is controversial and forall practical purposes is unknown. With the βarrestin knockout micedisclosed herein, it is shown that interfering with (eliminating) one ofthe key protein components of the desensitization mechanism greatlyenhances the potency and efficacy of the antinociceptive properties ofmorphine.

[0006] Accordingly, a first aspect of the present invention is aknockout mouse useful for testing the efficacy of potential analgesicagents, the cells of said mouse containing at least one inactiveendogenous βarrestin gene (preferably the βarrestin-2 gene), the mouseexhibiting a phenotype of decreased sensitivity to pain afteradministration of a μ opioid receptor agonist such as morphine ascompared to the corresponding wild type mouse. The mouse may beheterozygous or homozygous for the inactive endogenous βarrestin gene.The mouse is useful for evaluating potential analgesic drugs, andparticularly for evaluating the contribution of the desensitizationmechanisms to the antinociceptive effects of endogenous opioids.

[0007] A second aspect of the invention is a method of controlling painin a subject. The method comprises inhibiting βarrestin binding to thephosphorylated μ opioid receptor in said subject in an amount effectiveto induce or enhance analgesia in the subject. The method may be carriedout with or without concurrently administering a μ opioid receptoragonist (typically an opiate such as morphine) to said subject.

[0008] A third aspect of the present invention is a method of screeninga compound for activity in potentiating μ opioid receptor agonistactivity (e.g., morphine activity). The method comprises determiningwhether or not the compound inhibits βarrestin binding to aphosphorylated μ opioid receptor. The inhibition of such binding by thecompound indicates the compound is active in potentiating μ opioidreceptor agonist activity.

[0009] A particular aspect of the present invention is a method ofscreening a compound for activity in controlling pain. The methodcomprises determining whether or not the compound inhibits βarrestinbinding to phosphorylated μ opioid receptor. The inhibition of suchbinding by the compound indicates the compound is active in controllingpain (i.e., is a candidate compound for controlling pain, and should besubjected to further screening and testing for pain control). Any degreeof inhibition may be examined, with greater inhibition of bindingindicating potentially greater activity of the compound being tested.

[0010] Further aspects of the present invention include compoundsproduced or identified by the methods described hereinabove andpharmaceutical formulations of the same, along with the use of suchcompounds for the preparation of a medicament for the potentiation ofthe activity of μ opioid receptor agonists such as morphine, and/or forthe control of pain, in a subject in need thereof, either alone or incombination with a μ opioid receptor agonist such as morphine.

[0011] The foregoing and other objects and aspects of the presentinvention are explained in detail in the drawings herein and thespecification set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1. Characteristics of the targeted disruption of the mouseβarrestin-2 (βarr2) gene.

[0013]FIG. 1A. Schematic diagrams of βarr2 gene (top), targeting vector(middle) and the homologous recombinant gene (bottom) (7). The arrowsindicate the translational start and stop sites. The black boxesindicate the exons. A 0.8 kb Bam HI-Hind III fragment was replaced withthe pGK-neo cassette such that the entire exon 2, encoding amino acids9-19, was deleted. Transcription of the neomycin-resistant gene opposedthat of the βarr2 gene. Both 5′ and 3′ external probes were used ingenotype screening. Restriction enzyme sites are as follows: B, Bam HI;N, Nco I; H, Hind III; R, Eco RI.

[0014]FIG. 1B. Southern blot analysis of genomic DNA from wild type(WT), heterozygous (+/−) and homozygous (−/−) mice. Tail DNA wasdigested with Bam HI and analyzed by Southern blotting with the 5′ probeas shown in (A). A 3.5-kb fragment is indicative of the βarr2 knock-out(KO) allele and a 3-kb fragment is indicative of the wild-type allele.

[0015]FIG. 1C. Protein immunoblot analysis of βarr2 expression in WT,βarr2 +/−, and βarr2-KO mice. Membranes were blotted for βarr1 (top) andβarr2 (bottom) protein expression. Each lane was loaded with 25 μgprotein derived from the same lysates of the indicated brain regions.

[0016]FIG. 2. Enhanced and prolonged morphine-induced antinociception inβarr2-KO mice. Antinociceptive responses were measured as hot plate (56°C.) response latency after morphine (10 mg/kg, s.c.) treatment. The“response” was defined by the animal either licking the fore- orhind-paws or flicking the hind-paws. In these studies, the mostprominent response was fore-paw licking. To avoid tissue damage theanimals were not exposed to the plate for more than 30 seconds. Data arereported as the percent of the maximal possible response time (30seconds) which was determined from each individual mouse's basalresponse, the response after drug treatment, and the imposed maximumcutoff time with the following calculation (F. Porreca et al., JPharmacol Exp Ther 230, 341 (1984); J. Belknap et al., Physiol Behav 46,69 (1989). M. Gardmark et al., Pharmacol Toxicol 83, 252 (1998); G.Elmer et al., Pain 75, 129 (1998)): 100%×[(Drug response time−Basalresponse time)/(30 sec−Basal response time)]=% maximum possible effect(% MPE). WT (n=6), heterozygotes (+/−, n=5) and KO (n=9) mice wereanalyzed together in the same experiment. The % MPE curves of theβarr2-KO and βarr2 +/− mice were significantly greater than the WTresponse curve (P<0.001) as determined by two-way ANOVA.

[0017]FIG. 3. Greater dose-dependent antinociceptive responses tomorphine in βarr2-KO mice. The degree of antinociception was determinedby measuring latency of hot plate (56° C.) responses (FIG. 2).Withdrawal latencies were measured 30 min. after a first dose ofmorphine (1 mg/kg, s.c.) at which point, animals were immediatelyinjected with 4 mg/kg, s.c. morphine for a cumulative dose of 5 mg/kg.Antinociception was again assessed after 30 min. and mice wereimmediately injected with morphine (5 mg/kg, s.c.), to give a finalcumulative dose of 10 mg/kg. Withdrawal latencies were again measuredafter 30 min. after which, mice were immediately injected with naloxone(2.5 mg/kg, s.c.). After 10 min., antinociception was assessed oncemore. WT (n=7) and βarr2-KO (n=6) mice were significantly different ateach dose (*P<0.01, **P<0.001; Student's t-test). Means±S.E.M. areshown. In an additional experiment, morphine (25 mg/kg, s.c.) inducedthe maximum imposed response (100%) in both genotypes. Thus, anapproximate 2 fold shift in the apparent ED₅₀ values was observedbetween genotypes [WT: 9.77 (8.08-11.81) mg/kg; KO: 5.98 (5.10-6.94)mg/kg (95% confidence intervals)].

[0018]FIG. 4. Increased hypothermic responses to morphine in βarr2-KOmice. Rectal body temperatures were measured with a digital thermometer(M. Adler et al., Annu Rev Pharmacol Toxicol 28, 429 (1988); F.Fumagalli et al., J Neurosci 18, 4861 (1998) (TH8, Physitemp, Clifton,N.J., U.S.A.). The probe was inserted into the rectum and maintaineduntil the temperature reading stabilized. Basal body temperatures didnot vary significantly between genotypes (WT: 36.4±0.1° C.; KO:36.8±0.1° C). WT (n=5) and KO (n=5) animals were analyzed in parallelduring the same experiment. The curves are significantly different(P<0.001) as determined by 2-way ANOVA. Means±S.E.M. are shown.

[0019]FIG. 5. Binding of [³⁵S]GTPγS to periaqueductal gray membranesfrom βarr2-KO and wild type (WT) mice. [³⁵S]GTPγS binding to isolatedperiaqueductal gray (PAG) membranes (prepared as described inconjunction with Table 1 below) was determined after 2 hour stimulation(30° C.) with 50-10,000 nM of the mOR-selective agonist, [D-Ala2,MePhe4, Gly5-ol]enkephalin (DAMGO). PAG membranes (10 μg protein perassay tube) were incubated in the presence of 10 μM GDP and 50 pM[³⁵S]GTPγS (1250 Ci/mmol, NEN, Boston, Mass.). [³⁵S]GTPγS binding wasmeasured as described (P. Portoghese, in Handbook of ExperimentalPharmacology: Opioids I, A. Herz, Ed. (Springer-Verlag, N.Y., 1993) p.p.279-293. A. et al., ibid., p.p. 645-679). [³⁵S]GTPγS binding isexpressed as percent increase in [³⁵S]GTPγS binding relative to bindingin unstimulated samples. Data were analyzed by nonlinear regressionusing GraphPad Prism software and are presented as the mean±S.E.M of atleast three experiments performed in triplicate wherein WT and βarr2-KObrain regions were assayed simultaneously. In the absence of agoniststimulation, basal [³⁵S]GTPγS binding was: WT: 440±83 cpm and βarr2-KO:527±99 cpm.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The term “arrestin” as used herein has its ordinary meaning inthe art and is intended to encompass all types of arrestin, includingbut not limited to visual arrestin (sometimes referred to as Arrestin1), βarrestin 1 (sometimes referred to as Arrestin 2), and βarrestin 2(sometimes referred to as Arrestin 3).

[0021] The term “βarrestin” (or “β-arrestin”) as used herein is intendedto encompass all types of βarrestin, including but not limited toβarrestin 1 and βarrestin 2.

[0022] The phrases “concurrent administration,” “administration incombination,” “simultaneous administration” or “administeredsimultaneously” as used herein, interchangeably mean that the compoundsare administered at the same point in time or immediately following oneanother. In the latter case, the two compounds are administered at timessufficiently close that the results observed are indistinguishable fromthose achieved when the compounds are administered at the same point intime.

[0023] The production of βarrestin knockout mice can be carried out inview of the disclosure provided herein and in light of techniques knownto those skilled in the art, such as described in U.S. Pat. Nos.5,767,337 to Roses et al.; 5,569,827 to Kessous-Elbaz et al.; and5,569,824 to Donehower et al. (the disclosures of which applicantsspecifically intend to be incorporated by reference herein in theirentirety); and A. Harada et al., Nature 369, 488 (1994). Particularlypreferred mice for carrying out the present invention are also disclosedbelow.

[0024] 1. Assay Techniques

[0025] The step of determining whether or not βarrestin binding to thephosphorylated μ opioid receptor is inhibited by the test compound maybe carried out by any suitable technique, including in vitro assay andin vivo assay (e.g., in a cell that contains the βarrestin and thephosphorylated μ opioid receptor). A particularly suitable technique forin vivo assay is disclosed in U.S. Pat. No. 5,891,646 to Barak et al.(the disclosure of which is to be incorporated by reference herein inits entirety). In general, this technique involves providing a cell thatexpresses μ opioid receptor as a G-protein coupled receptor, andcontains the βarrestin protein conjugated to an optically detectablemolecule (e.g., green fluorescent protein). The test compound is thenintroduced into the cell (e.g., by microinjection, by electroporation,by suspending the cell in an aqueous solution that contains the testcompound, by contacting the cell to liposomes that contain the testcompound, by insertion of a heterologous nucleic acid into the cell thatencodes and expresses the test compound, etc.). Translocation of themolecule from the cytosol of the cell to the membrane edge of the cellis then monitored or examined, with the inhibition of such translocationindicating that the test compound inhibits the binding of βarrestin tothe phosphorylated μ opioid receptor. If desired, phosphorylation of theμ opioid receptor can be induced or enhanced by any suitable means, suchas contacting a μ opioid receptor agonist such as morphine to the cellin an amount effective to induce phosphorylation (e.g., by adding theagonist to the culture medium or liquid medium in which the cell iscontained). The cell is preferably a mammalian cell, but any suitablecell can be employed, including bacterial cells, yeast cells, fungalcells, plant cells, and other animal cells, so long as they express μopioid receptor and phosphorylate, or can be induced to phosphorylate,the same, and contain the desired βarrestin protein coupled to anoptically detectable molecule (e.g., either by exogenous introduction orexpression of the βarrestin conjugate therein). Any suitable βarrestinmay be employed as described above, with βarrestin-2 being preferred.

[0026] 2. Test Compounds

[0027] The present invention can be used with test compounds (or “probemolecules”), or libraries (where groups of different probe molecules areemployed), of any type. In general, such probe molecules are organiccompounds, including but not limited to oligomers, non-oligomers, orcombinations thereof. Non-oligomers include a wide variety of organicmolecules, such as heterocyclics, aromatics, alicyclics, aliphatics andcombinations thereof, comprising steroids, antibiotics, enzymeinhibitors, ligands, hormones, drugs, alkaloids, opioids,benzodiazepenes, terpenes, porphyrins, toxins, catalysts, as well ascombinations thereof. Oligomers include peptides (that is,oligopeptides) and proteins, oligonucleotides (the term oligonucleotidealso referred to simply as “nucleotide”, herein) such as DNA and RNA,oligosaccharides, polylipids, polyesters, polyamides, polyurethanes,polyureas, polyethers, poly (phosphorus derivatives) such as phosphates,phosphonates, phosphoramides, phosphonamides, phosphites,phosphinamides, etc., poly (sulfur derivatives) such as sulfones,sulfonates, sulfites, sulfonamides, sulfenamides, etc., where for thephosphorous and sulfur derivatives the indicated heteroatom for the mostpart will be bonded to C, H, N, O or S, and combinations thereof.Numerous methods of synthesizing or applying such probe molecules onsolid supports (where the probe molecule may be either covalently ornon-covalently bound to the solid support) are known, and such probemolecules can be made in accordance with procedures known to thoseskilled in the art. See, e.g., U.S. Pat. No. 5,565,324 to Still et al.,U.S. Pat. No. 5,284,514 to Ellman et al., U.S. Pat. No. 5,445,934 toFodor et al. (the disclosures of all United States patents cited hereinare to be incorporated herein by reference in their entirety).

[0028] 3. Pain Control and Active Compounds

[0029] As noted above, the present invention provides a method ofcontrolling pain in a subject, comprising inhibiting βarrestin bindingto the phosphorylated μ opioid receptor in said subject in an amounteffective to induce or enhance analgesia in the subject. The method maybe carried out with or without concurrently administering a μ opioidreceptor agonist such as morphine (or other opiate, as described below).When carried out without concurrent administration of μ opioid receptor,the analgesic activity relies upon the activity of endogenous opioidreceptor agonists.

[0030] The inhibiting of βarrestin binding (preferably βarrestin-2binding) to phosphorylated μ opioid receptor can be carried out directlyor indirectly by any suitable means, including but not limited toknockout of the βarrestin gene as described herein, disabling ordownregulating the kinase responsible for phosphorylation of the μopioid receptor, administration of an antisense oligonucleotide thatdownregulates expression of the βarrestin, or the administration of anactive compound that competitively inhibits binding of the βarrestin tophosphorylated μ opioid receptor (which may be identified by the assaytechniques described above). Obviously, functional μ opioid receptoritself must remain in the cells (particularly nerve cells) of thesubject so that the primary analgesic activity of the μ opioid receptoragonist can be exerted.

[0031] Compounds produced or identified as active compounds byapplication of the assay procedures described herein to the testcompounds or probe molecules described herein are useful in vitro and invivo as μ opioid receptor agonists (in that they enhance the activity ofopioids, although they do not bind to the same site as an opioid), areuseful in enhancing the efficacy, potency, or analgesic activity of μopioid receptor agonists. Such compounds are also useful in vivo incontrolling pain in a subject in need thereof. By “controlling pain”,“control of pain” and the like herein is meant partially or completelyinhibiting a pain response or perception of pain in a subject, and/orpartially or fully inducing local or general analgesia in a subject,either alone or in combination with another active agent administered tothe subject such as a μ opioid receptor agonist (e.g., morphine).Subjects that may be treated by the compounds identified by the presentinvention include both human subjects and animal subjects (e.g., dogs,cats, horses, cattle) for veterinary purposes.

[0032] Thus, as noted above, further aspects of the present inventioninclude active compounds produced or identified by the methods describedhereinabove and pharmaceutical formulations of the same (e.g., saidcompound in a sterile pyrogen-free saline solution), along with the useof such compounds for the preparation of a medicament for thepotentiation of the activity of μ opioid receptor agonists such asmorphine, and/or for the control of pain, in a subject in need thereof,either alone or in combination with a μ opioid receptor agonist such asmorphine.

[0033] In addition to morphine, other μ opioid receptor agonists,typically opiates, that may be used in conjunction with the presentinvention include, but are not limited to, codeine, oxycodeine,hydromorphone, diamorphine, methadone, fentanyl, sufentanil,buprenorphine, meperidine (Demerol®), etc.

[0034] The active compounds described above may be combined with apharmaceutical carrier in accordance with known techniques to provide apharmaceutical formulation useful carrying out the methods describedabove. See, e.g., Remington, The Science And Practice of Pharmacy (9thEd, 1995). In the manufacture of a pharmaceutical formulation accordingto the invention, the active compound (including the physiologicallyacceptable salts thereof) is typically admixed with, inter alia, anacceptable carrier. The carrier must, of course, be acceptable in thesense of being compatible with any other ingredients in the formulationand must not be deleterious to the patient. The carrier may be a solidor a liquid, or both, and is preferably formulated with the compound asa unit-dose formulation, for example, a tablet, which may contain from0.01 or 0.5% to 95% or 99% by weight of the active compound. One or moreactive compounds may be incorporated in the formulations of theinvention, which may be prepared by any of the well known techniques ofpharmacy consisting essentially of admixing the components, optionallyincluding one or more accessory ingredients.

[0035] The formulations of the invention include those suitable fororal, rectal, topical, buccal (e.g., sub-lingual), parenteral (e.g.,subcutaneous, intramuscular, intradermal, or intravenous), topical(i.e., both skin and mucosal surfaces), the most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used.

[0036] Formulations of the present invention suitable for parenteraladministration comprise sterile aqueous and non-aqueous injectionsolutions of the active compound, which preparations are preferablyisotonic with the blood of the intended recipient. These preparationsmay contain anti-oxidants, buffers, bacteriostats and solutes whichrender the formulation isotonic with the blood of the intendedrecipient. Aqueous and non-aqueous sterile suspensions may includesuspending agents and thickening agents. The formulations may bepresented in unit\dose or multi-dose containers, for example sealedampoules and vials, and may be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, saline or water-for-injection immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the kind previously described.For example, in one aspect of the present invention, there is providedan injectable, stable, sterile composition comprising a compound ofFormula (I), or a salt thereof, in a unit dosage form in a sealedcontainer. The compound or salt is provided in the form of alyophilizate which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form a liquid compositionsuitable for injection thereof into a subject. The unit dosage formtypically comprises from about 10 mg to about 10 grams of the compoundor salt. When the compound or salt is substantially water-insoluble, asufficient amount of emulsifying agent which is physiologicallyacceptable may be employed in sufficient quantity to emulsify thecompound or salt in an aqueous carrier. One such useful emulsifyingagent is phosphatidyl choline.

[0037] Formulations suitable for topical application to the skinpreferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol, or oil. Carriers which may be used include petroleumjelly, lanoline, polyethylene glycols, alcohols, transdermal enhancers,and combinations of two or more thereof.

[0038] Formulations suitable for transdermal administration may bepresented as discrete patches adapted to remain in intimate contact withthe epidermis of the recipient for a prolonged period of time.Formulations suitable for transdermal administration may also bedelivered by iontophoresis (see, for example, Pharmaceutical Research 3(6):318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the active compound. Suitable formulations comprisecitrate or bis\tris buffer (pH 6) or ethanol/water and contain from 0.1to 0.2M active ingredient.

[0039] Formulations suitable for oral administration may be presented indiscrete units, such as capsules, cachets, lozenges, or tablets, eachcontaining a predetermined amount of the active compound; as a powder orgranules; as a solution or a suspension in an aqueous or non-aqueousliquid; or as an oil-in-water or water-in-oil emulsion. Suchformulations may be prepared by any suitable method of pharmacy whichincludes the step of bringing into association the active compound and asuitable carrier (which may contain one or more accessory ingredients asnoted above). In general, the formulations of the invention are preparedby uniformly and intimately admixing the active compound with a liquidor finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet may be prepared bycompressing or molding a powder or granules containing the activecompound, optionally with one or more accessory ingredients. Compressedtablets may be prepared by compressing, in a suitable machine, thecompound in a free-flowing form, such as a powder or granules optionallymixed with a binder, lubricant, inert diluent, and/or surfaceactive/dispersing agent(s). Molded tablets may be made by molding, in asuitable machine, the powdered compound moistened with an inert liquidbinder.

[0040] The present invention is explained in greater detail in thefollowing non-limiting Examples.

EXAMPLE 1 Production of βArrestin Knockout Mice

[0041] Because GPCRs, such as the substance P receptor and the opioidreceptors, participate in processing the sensation of pain, wecharacterized analgesic responses through the μ opioid receptor (μOR) inmice lacking βarrestin-2. In the clinical setting, morphine is currentlythe most effective drug for alleviating intense and chronic pain. Theantinociceptive (blocking of pain perception) actions of morphine aremediated through stimulation of the μOR, as demonstrated by the lack ofmorphine analgesia observed in knock out mice deficient in the μOR (H.Matthes et al., Nature 383, 819 (1996). B. Kieffer, Trends Pharmacol Sci20, 19 (1999); I. Sora et al., Proc Natl Acad Sci USA 94, 1544 (1997)).Nevertheless, the neuronal signaling mechanisms mediating analgesiathrough μORs and morphine remain poorly understood. Moreover, thecontribution of GPCR desensitization to the onset and duration ofanalgesia has been unclear.

[0042] βarrestin-2 knockout (βarr2-KO) mice were generated byinactivation of the gene by homologous recombination. A bacteriophage λlibrary of mouse 129SvJ genomic DNA (Stratagene, La Jolla, Calif.) wasscreened with the rat βarr2 cDNA (H. Attramadal et al., J. Biol. Chem.267, 17882 (1992)). Positive phages were identified and analyzed byrestriction digest. A 12-kb βarr2 fragment was digested with Bam HI,subcloned into pBluescript KS(−) and sequenced. The targeting vector wasassembled by blunt-end ligation of a pHSV-TK cassette (frompIC19R/MCI-TK, M. R. Capecchi, University of Utah), a 2.8-kb Nco I-BamHI βarr2 fragment, a pGK-neo cassette (from plasmid pD383, R. Hen,Columbia University) which replaced the 0.8-kb Bam HI-Hind III fragmentof βarr2, and a 4.5 kb Hind III βarr2 fragment into pBluescript KS(−).This targeting vector was linearized with Not I and was electroporatedinto mouse embryonic stem cells. Genomic DNA from transfectantsresistant to G418 and gancyclovir were isolated and screened by Southern(DNA) blot analysis using a 0.2 kb 5′ external βarr2 probe and a 0.3 kb3′ external βarr2 probe. Chimeric animals were generated bymicroinjecting these ES cells into C57BL/6 blastocysts. Five chimericmale pups were obtained and mated with C57BL/6 females. Germlinetransmission was confirmed by Southern blotting. Heterozygous offspringwere intercrossed to obtain homozygous mice. Wild-type and mutant miceused in this study were age-matched, 3 to 5 month old, male siblings.For protein immunoblot analysis, whole cell lysates were prepared bypolytron homogenization in lysing buffer [10 mM Tris (pH 7.4), 5 mMEDTA, 1 protease inhibitor tablet/ 10 mL (Roche Molecular Biochemicals,Indianapolis, Ind., U.S.A.), 1% nonidet-40]. Polyacrylamide gels wereloaded with 25 μg protein/ lane and equivalent protein loading wasconfirmed by Ponceau S staining of the gels. After transfer topolyvinyldifluoride (PVDF) membranes, proteins were blotted withpolyclonal antibodies to βarrestin-2 or βarrestin-1 [H. Attramadal etal., J Biol. Chem. 267, 17882 (1992)]. Bands were visualized withsecondary antibody conjugated to horseradish peroxidase and an enhancedchemiluminescence detection system (Amersham, Piscataway, N.J.). Allexperiments were conducted in accordance with the NIH guidelines for thecare and use of animals.

EXAMPLE 2 Identification of βArrestin Knockout Mice

[0043] Mice lacking βarrestin-2 were identified by Southern DNA blotanalysis (FIG. 1A) and the absence of βarrestin-2 was confirmed byprotein immunoblotting of extracts from brainstem, periaqueductal gray(PAG) tissue, spleen, lung and skin (FIG. 1B). Wild-type, heterozygous,and homozygous mutant mice had similar amounts of βarrestin-1 in thebrain regions examined (FIG. 1B), arguing against compensatoryup-regulation of βarrestin-1 in the absence of βarrestin-2. The βarr2-KOmice were viable and had no gross phenotypic abnormalities. However,after administration of morphine, obvious differences became apparentbetween the genotypes.

EXAMPLE 3 Evaluation of Morphine-Induced Antinociception in βarrestinKnockout Mice

[0044] Morphine-induced antinociception was evaluated by measuringresponse latencies in the hot plate test. We used a dose of morphine (10mg/kg) and route of administration (s.c.) well established to induceanalgesia in many strains of mice (F. Porreca et al., J Pharmacol ExpTher 230, 341 (1984). J. Belknap et al., Physiol Behav 46, 69 (1989). M.Gardmark et al., Pharmacol Toxicol 83, 252 (1998). G. Elmer et al., Pain75, 129 (1998)). The analgesic effect of morphine was significantlypotentiated and prolonged in the knockout mice as compared to that intheir wild-type littermates (FIG. 2). Such robust responses to morphinewere not only absent in the wild-type littermates (FIG. 2) but also inthe parental mouse strains (C57BL/6 and 129SvJ) used to generate thisknockout. Four hours after the morphine injection, βarr2-KO mice stillexhibited significant analgesia (% maximum possible effect=31±0.4%);whereas, in control wild-type littermates, the analgesic effects of thesame dose of morphine waned after about 90 minutes. βarr2 +/− mice werenearly as responsive to morphine as the βarr2-KO mice; however, this mayreflect the imposed limit of the hot plate assay (30 seconds), which isdesigned to prevent prolonged exposure of the mice to pain. Basalresponses to the hot plate did not differ between genotypes (wild type:6.2±0.3 sec., n=25; βarr2-KO: 6.1±0.4sec., n=27). The differences inmorphine-induced analgesia between the genotypes are unlikely to be dueto pharmacokinetic differences in morphine metabolism, because theconcentrations of morphine in blood, as determined by mass spectroscopyanalysis, did not differ between wild type and βarr2-KO mice 2 hoursafter morphine injection (Mice were injected with morphine (10 mg/kg,subcutaneous). After 30 minutes or 2 hours, wild-type mice were killedand blood was collected in vials containing sodium-fluoride andpotassium-oxalate. Morphine concentration in blood samples pooled from 3mice per sample were 1,500 ng/mL after 30 min., and 83 ng/mL blood after2 hours as measured by mass spectroscopy analysis [Occupational TestingDivision of LabCorp, Inc., Research Triangle Park, N.C., U.S.A.]. Insimilar experiments, βarr2-KO mice had a concentration of 93 ng/mL inthe blood after 2 hours).

EXAMPLE 4 Evaluation of Low Dosage Morphine in βarrestin Knockout Mice

[0045] Lower doses of morphine were also tested in these assays. Even atdoses of morphine (1 mg/kg, s.c.) that were sub-analgesic in wild typemice, βarr2-KO animals displayed a significant increase in theirnociceptive thresholds (FIG. 3). At 30 minute intervals, immediatelyfollowing the antinociception test, mice were given repeated cumulativedoses of morphine resulting in final concentrations of 5, and 10 mg/kg(I. Sora et al., Proc Natl Acad Sci USA 94, 1544 (1997)). At the highestcumulative dose, mice reached similar levels of antinociception as seenin FIG. 2, in which this amount of morphine was administered in a singleinjection. At every dose, the βarr2-KO animals experienced greaterantinociception after morphine treatment than did their wild-typelittermates.

EXAMPLE 5 Evaluation of Morphine Antagonists in βarrestin Knockout Mice

[0046] To test whether the analgesic effects of morphine were mediatedby actions at the μOR, mice were treated with various antagonists.Naloxone (2.5 mg/kg, subcutaneous injection) which immediately reversesthe effects of opiates, was given 30 minutes after morphine (10 mg/kg).Naltrindole [P. Portoghese et al., J Med. Chem. 88, 1547 (1990)] wasgiven 20 minutes before morphine, and nor-binaltorphimine (A. Takemoriet al., J Pharmacol Exp Ther 246, 255 (1988)) was given 1 hour beforemorphine (H. Matthes et al., J Neurosci 18, 7285 (1998)).

[0047] Naloxone, a well-established OR antagonist, was administered tothe same mice, immediately after measuring the antinociceptive effectsof morphine (10 mg/kg). Naloxone (2.5 mg/kg, s.c.) completely reversedthe effects of morphine in both the wild-type and βarr2-KO animalswithin 10 minutes. However, the δ and κ OR-selective antagonistsnaltrindole (2.5 mg/kg, s.c.) and nor-binaltorphimine (5 mg/kg s.c.) didnot inhibit analgesia in wild type nor βarr2-KO mice (data not shown).The morphine dose dependency of the antinociceptive response and thereversal of the effects with naloxone suggest that the potentiated andprolonged effects in mice that lack βarrestin-2 result from stimulationof the μOR.

EXAMPLE 6 Body Temperature Measurements in Wild-Type and βarrestinKnockout Mice

[0048] Wild-type and βarr2-KO mice were also evaluated for changes inbody temperature (M. Adler et al., Annu Rev Pharmacol Toxicol 28, 429(1988). Rectal body temperatures were determined with a digitalthermometer [F. Fumagalli et al., J Neurosci 18, 4861 (1998)] (TH8,Physitemp, Clifton, N.J., U.S.A.). The probe was inserted into therectum and maintained until the temperature reading stabilized). Nosignificant differences in basal body temperature were found betweengenotypes, however βarr2-KO mice experienced a greater drop in bodytemperature after morphine treatment than did wild-type (FIG. 4). Thisgreater decrease in temperature also persisted longer than that in theirwild type littermate controls.

EXAMPLE 7 Radioligand Binding Assays

[0049] To investigate whether the μOR population was altered in the KOmice, radioligand binding analysis on membranes prepared from differentbrain regions was performed.

[0050] Brain regions were dissected and immediately frozen in liquidnitrogen and were stored at −80° C. for less than 1 week before use.Samples were placed on ice and homogenized by polytron in membranepreparation buffer [50 mM Tris (pH 7.4), 1 mM EDTA, 3 mM MgCl₂] andcrude membranes were prepared by centrifugation at 20,000×g for 15 minat 4° C. Membranes were resuspended in either 50 mM Tris-HCl (pH 7.4)for radioligand binding assays or in assay buffer [50 mM Tris-HCl (pH7.4), 100 mM NaCl, 3 mM MgCl2, 0.2mM EDTA] containing 10 μM GDP for[³⁵S]GTPγS binding assays. For both binding assays, reactions wereterminated by rapid filtration over GF/B filters (Brandel, Inc.,Gaithersburg, Md.) using a Brandel cell harvester (Brandel, Inc.,Gaithersburg, Md.). Filters were washed 3 times with ice cold 10 mMTris-HCl (pH 7.4) and then counted in a liquid scintillation counter.Hypothalamus, brain stem, and periaqueductal gray (PAG) regions werechosen because they contain μORs and are implicated in the regulation ofpain and body temperature (D. Mayer and D. Price, Pain 2, 379 (1976). T.Yaksh et al., Prog Brain Res 77, 371 (1988). D. J. Smith, et al., Eur JPharmacol 156, 47 (1988)). Data are given in Table 1. Saturation bindingstudies with ³H-naloxone, at concentrations that preferentially labelthe μOR, revealed a single high affinity binding site, which representsthe μOR. The number and affinity of μORs did not significantly differbetween the two genotypes in any of the brain regions examined. TABLE 1³H-Naloxone binding in brain regions of Wild Type and Knockout mice.¹Wild Type βarr2-Knockout B_(MAX) K_(D) B_(MAX) K_(D) Brain Region(fmol/mg) (nM) (fmol/mg) (nM) PAG 132 ± 9  4.0 ± 0.1 144 ± 13 4.5 ± 0.8Brainstem 49 ± 7 1.5 ± 0.2 54 ± 9 3.0 ± 0.8 Hpothalamus 103 ± 18 6.2 ±1.6 89 ± 8 3.8 ± 0.2 # duplicate.

[0051] Additional evidence for increased sensitivity of the μOR inβarr2-KO animals was obtained in biochemical experiments. We measuredagonist-stimulated binding of [³⁵S]GTPγS to G proteins in isolatedmembranes the most proximal manifestation of GPCR activation (D. Selleyet al., Mol Pharmacol 51, 87 (1997)). Because morphine acts in vitro tostimulate μ, δ, and κ opioid receptors, the μOR-selective agonist,[D-Ala², MePhe⁴, Gly⁵-ol]lenkephalin (DAMGO), was used to specificallyactivate G protein coupling to μORs. DAMGO stimulated more [³⁵S]GTPγSbinding in membranes derived from βarr2-KO mice than in those derivedfrom wild-type littermates (FIG. 5). Similar results were also obtainedin brainstem membranes (data not shown). The amount of Gα proteins(G_(i/o/z)) as determined by protein immunoblotting, did not varybetween the genotypes (data not shown). These observations suggest thatthere is enhanced coupling of μORs to G proteins in tissues derived fromβarr2-KO mice. Although the enhanced analgesia induced by morphine mayinvolve complex neurological signaling, this biochemical evidencesupports the interpretation that the enhanced physiologicalresponsiveness in the knockout animals results from increasedsensitivity of signaling by the μOR.

[0052] These studies demonstrate in an animal model that the absence ofβarrestin-2 can affect the efficacy of GPCR activation. In transfectedcultured cells, the degree of β₂-adrenergic receptor signaling isdependent upon the cellular complement of GRK2 and GRK3 and βarrestins(L. Menard et al., Mol Pharmacol 51, 800 (1997); S. Mundell et al.,Biochemistry 38, 8723 (1999)). These observations, along with thosepresented here, directly support the proposed role of βarrestin-2 inpreventing further receptor-G protein coupling and mediatingdesensitization of the GPCR. Moreover, βarrestins are not only involvedin the dampening of GPCR responsiveness after agonist stimulation, butalso influence the sensitivity of the response.

[0053] The simplest interpretation of these results is that μORsignaling is regulated by βarrestin-2. However, in transfected cells,morphine fails to induce the internalization of the μOR and a GFP-taggedβarrestin-2 fails to translocate to μOR overexpressed in cell cultureupon exposure to morphine (J. Arden et al., J Neurochem 65, 1636 (1995).D. Keith et al., J Biol Chem 271, 19021 (1996); J. Whistler and M. vonZastrow, Proc Natl Acad Sci U S A 95, 9914 (1998); J. Zhang et al., ProcNatl Acad Sci USA 95, 7157 (1998)). Interestingly, these in vitrostudies have been conducted with the rat μOR or the mouse MOR1 which arenot particularly rich in potential phosphorylation sites. Several splicevariants of the μOR are present in mouse brain that contain severalpotential phosphorylation sites (Y. Pan et al., Mol Pharmacol 56, 396(1999)). Some of these isoforms can contribute to morphine-inducedanalgesia. The involvement of these receptors might explain thedifferences between the in vitro studies and those with the βarr2-KOmice.

[0054] The βarr2-KO mice were very similar in phenotype to their wildtype littermates and other GPCR-directed drugs did not necessarilyelicit different responses between the genotypes. For example, locomotorresponses to dopamine receptor stimulation by cocaine and apomorphinewere not enhanced (data not I shown). These observations suggest thatvarious GPCRs are differentially affected by the loss of βarrestin-2.Other regulatory elements, such as GRKs or βarrestin-1, could compensatefor the lack of βarrestin-2, or the receptors could vary in theirrequirement for βarrestin interaction for their regulation.

[0055] The foregoing is illustrative of the present invention, and isnot to be construed as limiting thereof. The invention is defined by thefollowing claims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A knockout mouse useful for testing theefficacy of potential analgesic agents, the cells of said mousecontaining at least one inactive endogenous βarrestin-2 gene, said mouseexhibiting a phenotype of decreased sensitivity to pain afteradministration of a μ opioid receptor agonist as compared to thecorresponding wild type mouse.
 2. A knockout mouse according to claim 1,wherein said mouse is heterozygous for an inactive endogenousβarrestin-2 gene.
 3. A knockout mouse according to claim 1, wherein saidmouse is homozygous for an inactive endogenous βarrestin 2 gene.
 4. Amethod of controlling pain in a subject, comprising inhibiting βarrestinbinding to the phosphorylated μ opioid receptors in said subject in anamount effective to induce or enhance analgesia in said subject.
 5. Amethod according to claim 4, further comprising the step of concurrentlyadministering a μ opioid receptor agonist to said subject.
 6. A methodaccording to claim 5, wherein said μ opioid receptor agonist is selectedfrom the group consisting of morphine, codeine, oxycodeine,hydromorphone, diamorphine, methadone, fentanyl, sufentanil,buprenorphine, and meperidine.
 7. A method according to claim 5, whereinsaid μ opioid receptor agonist is morphine.
 8. A method of screening acompound for activity in controlling pain, comprising: determiningwhether or not said compound inhibits βarrestin binding to aphosphorylated μ opioid receptor; the inhibition of such binding by saidcompound indicating said compound may be active in controlling pain. 9.A method according to claim 8, wherein said determining step is carriedout in vitro.
 10. A method according to claim 8, wherein said βarrestinis βarrestin
 2. 11. A method of screening a compound for activity inpotentiating μ opioid receptor agonist activity, comprising: determiningwhether or not said compound inhibits βarrestin binding to aphosphorylated μ opioid receptor; the inhibition of such binding by saidcompound indicating said compound is active in potentiating μ opioidreceptor agonist activity.
 12. A method according to claim 11, whereinsaid determining step is carried out in vitro.
 13. A method according toclaim 11, wherein said βarrestin is βarrestin
 2. 14. A method accordingto claim 11, wherein said μ opioid receptor agonist is selected from thegroup consisting of morphine, codeine, oxycodeine, hydromorphone,diamorphine, methadone, fentanyl, sufentanil, buprenorphine, meperidine.15. A method according to claim 11, wherein said μ opioid receptoragonist is morphine.